The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine of molecular weight greater than one billion Daltons. The PSD is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we developed method to freeze-substitute hippocampal cultures and then examine them in thin sections by EM tomography which show individual protein complexes in their natural setting within the PSD. The initial work employing tomography revealed that the core of the PSD is an array of vertically oriented filaments that contain the scaffold protein, PSD-95, in an extended configuration and a polarized orientation, with its N-terminus positioned at the postsynaptic membrane. This finding provides insight into the overall organization of the PSD because scaffolding proteins such as PSD-95 family MAGUK proteins have distinct multiple, diverse binding sites for other proteins arrayed along their length. Thus, the regular arrays of PSD-95 perhaps with other family members impose an ordering on many other PSD proteins, including the glutamate receptors, and provide an overall plan for the structure of the PSD. FRET constructs were made to study possible mechanisms that regulate PSD-95 MAGUK conformations in collaboration with the Green laboratory (U Chicago). Two fluorophores (RFP and YFP) are fused to the opposite termini of PSD-95 or other family members. The labels allow the conformations of the family members to be determined both by immunogold-EM and by making FRET measurements on living cells (if the two ends of the molecule FRET, they must be in a closed configuration). So far results suggests that PSD-95 adopts an extended conformation in PSDs but in closed conformation at non-synaptic sites. Open conformation of PSD-95 at the PSD is a requirement for it to interact with NMDAR and AMPAR-Stargazin complexes. N-terminal palmitoylation appeared to be a determinant in switching PSD-95 conformations. EM tomography also revealed that the C-terminal ends of the vertical filaments are associated with horizontally oriented filaments. One class of horizontal filament is ordered to form hexagonal cross-linkers with the vertical filaments, and is concentrated beneath the NMDA receptors. Immunogold labeling now tentatively identifies a class of horizontal filaments as GKAP, which is a known to bind to the GK domain at the C-terminal end of PSD-95. Immunogold labeling is also being used to locate another major scaffolding molecule, SHANK, which is known to bind GKAPs directly. The emerging structural model of the PSD shows how the PSD-95 matrix can stabilize glutamate receptors, and at the same time could be allow room for the addition of new receptors at the edges of the PSD. Identification of the components of the PSD is time consuming and the methods for identifying the proteins need improvement. An expressible probe, MiniSOG, has become available and we are having success using it to confirm that the vertical filaments are PSD-95. We now preparing probed to use MiniSOG to definitely identify GKAP and SHANK in the PSD. The idea that the PSD-95 dependent scaffold stabilizes the PSD has been explored by using EM tomography to determine the effects of RNAi knock down of PSD-95. After PSD-95 is knocked down, patches of PSD unravel, demonstrating the central role of the PSD-95 scaffold in organizing the PSD. Recently we examined the effects of knocking down simultaneously three major MAGUK proteins: PSD-95, PSD-93 and SAP102, and for the first time, EM tomography revealed significant loss from the central core of the PSD, including NMDA receptor structures, vertical filaments, and AMPA receptors. Electrophysiology measurements by collaborators from the Nicol laboratory (UCSF) characterizing the effects of the same knock down show significant functional loss of NMDAR and AMAPR type EPSPs at levels compatible with the structural losses. A newly developed electron microscopic method at Dr. Richard Leapmans lab (NIBIB) using high voltage STEM tomography (HVST) is compatible with sections up to two m thick and is revealing detailed reconstructions of many whole synapses. We used HVST on 1-2 um sections that contain entire PSDs at synapses to confirm that simultaneous knock down of the three major PSD-95 family MAGUKs results in significant reduction in the overall PSD area, leaving many synapses with only small PSDs, Again the structural loss correlates closely well with functional loss measured by physiological recordings. A method for high resolution EM tomography of isolated PSDs became available with the discovery of a negative stain compatible with tomography. Using this stain makes it possible accurately to map the distribution of CaMKII in isolated PSDs. A second pool of CaMKII is found embedded in the matrix of the PSD, which may be functionally distinct from the soluble pool of CaMKII that binds to the PSD during activity. This pool has been invisible to light microscopic analyses and so revives the idea that the CaKII in the PSD may be the functionally important pool. These ideas will be tested directly by inducing different states of potentiation in cultured neurons and examining the distribution of CaMKII in their PSDs, and this project now becomes part of Project #NS003113-04, Dynamic Structural Properties of Synapses.